[CANCER RESEARCH 60, 712–721, February 1, 2000]

Integrins avb3 and avb5 Are Expressed by Endothelium of High-RiskNeuroblastoma and Their Inhibition Is Associated with IncreasedEndogenous Ceramide1

Anat Erdreich-Epstein, Hiroyuki Shimada, Susan Groshen, Ming Liu, Leonid S. Metelitsa, Kwang Sik Kim,Monique F. Stins, Robert C. Seeger, and Donald L. Durden2

Divisions of Hematology-Oncology [A. E-E., M. L., L. S. M., R. C. S., D. L. D.] and Infectious Diseases [K. S. K., M. F. S.], Neil Bogart Memorial Laboratories, and Departmentsof Pediatrics and Pathology [H. S.], Children’s Hospital Los Angeles, University of Southern California School of Medicine, Los Angeles, California 90027; and Department ofPreventive Medicine [S. G.], University of Southern California School of Medicine and the Children’s Cancer Group, Arcadia, California 91066

ABSTRACT

Inhibition of the RGD-binding integrins, avb3 and avb5, preventsendothelial cell anchorage and induces endothelial apoptosis, which re-sults in disruption of tumor angiogenesis and inhibition of tumor growthin animal models. In this study, we demonstrate by immunohistochemicalanalysis that integrin avb3 was expressed by 61% (mean) of microvesselsin high-risk neuroblastomas (stage IV and MYCN-amplified stage III;n 5 28) but only by 18% (mean) of microvessels in low-risk tumors (stagesI and II and non-MYCN-amplified stage III; n 5 12). Integrin avb5 wasfound on 60% (mean) of microvessels in 21 Stage IV tumors. These datasuggest that neuroblastomas may be targeted for antiangiogenic treatmentdirected against endothelial integrins avb3 and avb5. In cell culture,inhibition of integrin-dependent endothelial cell anchorage to vitronectinby RGDfV, an RGD function-blocking cyclic peptide, induced apoptosis inbovine brain endothelial cells compared with the control peptide, RADfV(37.5% versus8.7%, respectively), as detected by chromatin condensationand nuclear fragmentation. Treatment with RGDfV but not with RADfV,which prevented attachment of endothelial cells to vitronectin or fibronec-tin, was associated with up to a 50% increase in endogenous ceramide, alipid second messenger that can mediate cell death. Furthermore, exoge-nous C2-ceramide was cytotoxic to bovine brain endothelial cells andinduced activation of C-jun N-terminal kinase (JNK), a MAP kinase thatcan be activated in stress-induced apoptosis pathways. This suggests thatceramide may function in detachment-induced endothelial cell apoptosis,originating from inhibition of vitronectin binding to integrins such as avb3

and avb5. This is the first report to demonstrate expression of integrinsavb3 and avb5 by microvascular endothelium of a childhood tumor andassociation of their expression with neuroblastoma aggressiveness. Fur-thermore, our data provide the first suggestion that inhibition of endo-thelial cell anchorage, resulting from specific blockade of RGD-bindingintegrins, increases endogenous ceramide, which may contribute to endo-thelial cell death.

INTRODUCTION

Integrins area/b heterodimeric cell-surface receptors that link theextracellular matrix with the cell cytoskeleton and generate intracel-lular signals to regulate cell survival and growth, cell differentiation,and cell motility. Vitronectin, a matrix protein comprising two he-

mopexin domains, a somatomedin B domain and a cell-binding re-gion, binds integrinsavb1, avb3, avb5, avb6, avb8, aIIbb3, anda8b1

via the RGD sequence in the cell-binding region at its NH2-terminus(1–4). Integrin binding to vitronectin can be inhibited by specificfunction-blocking antibodies or by synthetic RGD peptides, whichmimic the conformation of the RGD sequence (5–8). Of the syntheticRGD peptides, some of the most active in inhibiting cell attachmentto vitronectin are the cyclic pentapeptides (5, 6). To date, the onlyvitronectin-binding integrins that have been described on the surfaceof endothelial cells areavb3, avb5 (7–9), and to a lesser extentavb6

(10).The angiogenic integrins,avb3 andavb5, which are expressed on

endothelial cells, are crucial for their survival (8, 11–14). Survivalsignals transmitted by integrinavb3 lead to inhibition of p53 activity,decreased expression of p21WAF1/CIP1, and suppression of the baxcell death pathway in endothelial cells (15). When plated on osteopon-tin, the avb3-dependent signals for endothelial cell survival are me-diated via NFkB3 (16). These survival pathways can be blocked eitherby function-blocking antibodies or by peptide analogues, which blockthe active RGD binding site on integrinsavb3 andavb5, thus inducingapoptosis of the angiogenic endothelial cells (8, 11, 12). Endothelialapoptosis results in inhibition of new blood vessel formation, disrup-tion of existing angiogenic vasculature, inhibition of tumor growth,and tumor regression (8, 11, 12, 14–16).

Apoptosis of endothelial cells can occur in response to a variety ofstress stimuli such as LPS (17), TNFa (18), ionizing radiation (19),growth factor withdrawal (20), and disruption of matrix binding orspecific integrin ligation (11, 21). Ceramide, a lipid second messengerthat is synthesizedde novoor derived from membrane sphingomyelin,is implicated in apoptotic signaling pathways induced by stimuli suchas irradiation, TNFa, and LPS (22–24) and has been linked to acti-vation of pro-apoptotic MAPKs (e.g.,JNK/SAPK; Ref. 23). Thedelicate balance between cellular levels of the survival-promoting,sphingosine-1-phosphate and the apoptosis-linked ceramide, both me-tabolites of sphingomyelin, provides a means for sensitive regulationof pro- and antiapoptotic MAPKs (e.g.,JNK/SAPK, p38, or extracel-lular regulated kinases), thus contributing to regulation of cell survivaland death (25). Interestingly, ceramide enhances expression ofMMP-13 in fibroblasts (26), suggesting that it may mediate signals formatrix remodeling and possibly for angiogenesis. Although ceramidehas been implicated in endothelial apoptosis induced by a variety ofstimuli bothin vivo (17) andin vitro (19, 23), it is not known whether

Received 4/19/99; accepted 12/2/99.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grant CA75637 to D.L.D. from the National Institutesof Health and by grants to A.E-E. from the Childrens Cancer Research Fund and theConcern Foundation. This work was also supported in part by Grant UO1, CA70903-02from NIH (to D. L. D.); Grants CA60104 and CA02649 from the National CancerInstitute, Department of Health and Human Services (to R. C. S.); Grant CA14089 (toS. G.); Grants NS26310 and HL61951 (to K. S. K.); the Neil Bogart Memorial Fund of theT.J. Martell Foundation for Leukemia, Cancer, and AIDS Research; “My Brother JoeyFoundation” founded by Judi and Art Partridge, and the Michael Hoefflin Foundation forChildren’s Cancer (to A. E-E.); and by the STOP Cancer Award (to D. L. D.).

2 To whom requests for reprints should be addressed, at the Department of Pediatrics,Herman B. Wells Center for Pediatric Research, Cancer Research Institute, 1044 WestWalnut Street, Room 468, Indiana University School of Medicine, Indianapolis, IN 46202.Phone: (317) 278-3718; Fax: (317) 274-8679; E-mail: ddurden@iupui.edu.

3 The abbreviations used are: NFkB, nuclear factorkB; LPS, lipopolysaccharide;TNFa, tumor necrosis factora; MAPK, mitogen-activated protein kinase; JNK, C-junN-terminal kinase; SAPK, stress-activated protein kinase; MMP, matrix metalloprotein-ase; NB, neuroblastoma; BBEC, bovine brain endothelial cells; mAb, monoclonalantibody; HUV-EC-C, human umbilical vein endothelial cells; MTT, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide; GST, glutathioneS-transferase; MKI,mitosis-karyorrhexis index; AKT/PKB, protein kinase B; BOC-D-FMK, BOC-aspar-tyl(OMe)-fluoromethylketone.

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ceramide plays a role in signaling of apoptosis resulting from inhibi-tion of integrin-mediated endothelial cell anchorage.

Treatment of NB, the second most common solid tumor in child-hood, is successful in less than half of patients with high-risk (stage IVand MYCN-amplified stage III) disease (27–29). A high vascularindex in NB correlates with poor prognosis (30), suggesting depend-ence of aggressive tumor growth on active angiogenesis, the sproutingof new capillaries from existing blood vessels. The matrix-degradingenzymes MMP-2 and MMP-9 are elevated in high-risk NB comparedwith low-risk disease (31) and are expressed in NB cell lines, whichthemselves induce proliferation of endothelial cells (32), supportingthe angiogenic phenotype of this tumor. Moreover, several antiangio-genic approaches have demonstrated activity in animal models of NB(33–35). Thus, utilization of antiangiogenic approaches, by them-selves or in combination with other therapies, could potentially im-prove the outcome in children with NB and may warrant further study.

In view of thein vivo efficacy of antiangiogenic treatment directedagainst integrinsavb3 andavb5 in animal models (8, 11, 12, 35) andthe correlation of a high vascular index with poor prognosis in NB(30), we hypothesized that tumor endothelium in NB may present apotential target for antiangiogenic treatment directed against integrinsavb3 and/or avb5. Furthermore, we hypothesized that ceramide,which can contribute to apoptotic signaling in endothelial cells, maybe involved in apoptosis induced by inhibition of integrin-mediatedanchorage in these cells. In this study, we report that inhibition ofendothelial cell attachment and spreading and the resultant apoptosis,originating from blockade of RGD-binding integrins such asavb3 andavb5, are associated with the generation of endogenous ceramide. Inaddition, we show by immunohistochemical analysis that integrinsavb3 andavb5 are expressed on average by 61% of microvessels inthe high-risk NB (stage IV and MYCN-amplified stage III;n 5 28)but avb3 is only expressed in 18% of low-risk tumors (stages I and IIand non-MYCN-amplified stage III;n 5 12). These data suggest thatNB endothelium may be targeted for antiangiogenic treatment di-rected against integrinsavb3 andavb5.

MATERIALS AND METHODS

Cells, Reagents, and Antibodies.All reagents were purchased from Sigma(St. Louis, MO) unless stated otherwise. BBEC transfected with large-Tantigen were maintained in RPMI 1640 supplemented with glutamine, pyru-vate, 10% FCS, and 10% Nu-Serum IV culture supplement (CollaborativeBiomedical Products, Bedford, MA) as described (36). These cells spontane-ously expressed high levels of integrinavb3 on their surface (mean, 92.4%,SD, 5.8%, by fluorescence-activated cell-sorting analysis using mAb LM609in 11 separate determinations over a period of at least 4 months in culture).Primary HUV-EC-C were purchased from the American Type Culture Col-lection (passage 16; Manassas, VA), maintained according to the AmericanType Culture Collection recommendations, and used between passages 16 and24. Previously characterized affinity-purified mAb to integrinavb3 (LM609)and theav integrin subunit (LM142) were a generous gift from D. Cheresh (7,12, 14). Monoclonal antibody clone P1F6 against integrinavb5 was purchasedfrom Life Technologies (Gaithersburg, MD). Isotype-specific mouse IgG1(control for the mAb stains) and polyclonal anti-Factor VIII were from Dako(Carpinteria, CA). Secondary antibodies for the double immunofluorescentstains were FITC-conjugated goat F(ab9)2 anti-rabbit IgG and rhodamine-conjugated goat F(ab9)2 anti-mouse IgG (BioSource International, Camarillo,CA). Human fibronectin (domains 2–11, constituting the 110-kDa cell-bindingfragment containing the RGD site but not the Hep-2/CS1 region) was pur-chased from Upstate Biotechnology (Lake Placid, NY), and human vitronectinwas purchased from Promega (Madison, WI) or prepared as described byYatohgo et al. (37). The cyclic pentapeptides RGDfV and RADfV weregenerous gifts from Merck (Darmstadt, Germany) via Alfred Jonczyk. All cellculture experiments were repeated at least three times.

Patients and Tumor Specimens.The NB specimens included in this studywere resected at institutions of the CCG (Arcadia, CA) between 1986 and1992. Clinical staging was performed according to standard criteria used by theCCG (38). Neuroblastoma tumor tissue was obtained surgically, snap-frozen,and shipped on dry ice to the CCG Neuroblastoma Biology Reference Labo-ratory. Upon receipt, a portion was removed without thawing, placed inTissue-Tek OCT embedding compound (Miles, Elkart, IN), and maintained at270°C. Another part of each tumor was fixed in formalin and embedded forlight microscopic examination. Histological sections of tumors were evaluatedfor stromal cells, neuroblastic differentiation, and mitotic/karyorrhectic cellnumber and categorized as having favorable or unfavorable histology (39).Forty-two tumors were analyzed. Two of the forty-two samples processed wereexcluded from the analysis after processing because during the pathologicalanalysis one sample was found to be a lymph node metastasis rather than asample from the primary tumor and one OCT block was inadequate (itcontained only tumor capsule). Patient and tumor characteristics for the re-maining 40 patients are summarized in Table 1. Thirty-five of the sampleswere obtained at the time of diagnosis, before the patient had received anychemotherapy or radiation; five of the samples were obtained from tumorsresected from patients who had already received chemotherapy (i.e.,at the timeof delayed resection or after relapse).

Immunohistochemistry. Immunohistochemistry was performed on 6-mmserial cryostat sections freshly cut from the preserved OCT blocks, which wereimmediately fixed in cold acetone (for 5 min) and air-dried. Because mAbLM609 (anti-integrinavb3) only recognizes the nondenatured functional con-formation of integrinavb3 (40), it was not possible to use paraffin blocks forthis study. All washes and dilutions of reagents were done in Tris-bufferedsaline (10 mM Tris-HCl (pH 7.6) and 130 mM NaCl) at room temperature.After three washes the sections were blocked for 5 min in 2% goat serum (LifeTechnologies); reacted with primary antibodies for 2 h (LM609, 1:500; P1F6,1:400; or Anti-Factor VIII, 1:1000); washed three times; incubated for 60 minwith multilink (swine) anti-goat, -mouse, and -rabbit immunoglobulins (1:50;Dako); washed again; incubated for 30 min with avidin-biotin-peroxidase(Vector Laboratories, Burlingame, CA); reacted for 10 min with 3,3-diamino-benzidine (0.4 mg/ml); rinsed; counter-stained with Mayer’s hematoxylin;mounted; and read. Mouse IgG1, used as a negative control, was negative inall cases.

Immunofluorescence Analysis.Immunofluorescent staining was per-formed as previously described with minor modifications (12). Four-mmcryostat NB sections in OCT were fixed in cold acetone for 30 s, air dried, andsubsequently blocked with 2.5% BSA in PBS (pH 7.4) for 1–2 h at roomtemperature. Sections were then washed 5–8 times in PBS and incubated withmonoclonal anti-avb3 (LM609 1:50, 60mg/ml) and polyclonal anti-Factor VIII(1:1000) for 1 h at room temperature. Sections were washed and then incubatedfor 1.5 h at room temperature with rhodamine-conjugated anti-mouse IgG(1:300) and FITC-conjugated anti-rabbit IgG (1:300). Tissue sections werethen washed, mounted, and photographed using an Olympus AX70 compoundmicroscope (Olympus America, Melville, NY) at3200.

Cell Radiolabeling and Analysis of Ceramide.Ceramide metabolism wasstudied as described (41–43). Briefly, endothelial cells were plated the daybefore the experiment (33 106 cells/100-mm plate, in their usual growthmedium) and metabolically labeled with 9,10 [3H]-(N)-palmitic acid (1mCi/ml; NEN, Boston, MA) overnight at 37°C. The next day the medium wascollected, and cells were briefly trypsinized, washed, and replated on non-tissue culture-treated 6-well plates that were precoated with fibronectin (10mg/ml), vitronectin (2mg/ml), or BSA (0.5%) and blocked with BSA (0.5%)in the medium in which they were incubated overnight (106 cells/well). Whereindicated, the cells were replated in the presence or absence of the cyclicRGD-blocking peptide RGDfV or control RADfV. At the end of incubation thecells were briefly trypsinized, combined with the nonadherent cells of the samewell, washed (at 4°C), and lipid was extracted using equal volumes of meth-anol/2% acetic acid (v/v), water, and chloroform. The dried lipid was solubi-lized in chloroform:methanol (2:1 v/v) and analyzed using TLC plates with asolvent system consisting of chloroform:acetic acid (9:1 v/v) and using lipidstandards as markers as described (41–43). Ceramide and total cellular lipidswere quantitated in a liquid scintillation counter (cpm), and ceramide wascalculated and expressed as percent [3H]ceramide of total [3H]-labeled cellularlipids extracted.

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Cell Density and Viability. Cell viability was assessed by the uptake ofMTT (Thiazolyl blue; Sigma), a process which is dependent on intact mito-chondrial function, as described by Mosmann (44). This method yields resultssimilar to those of other methods of assessing endothelial cell viability (45).Briefly, to assess viable cell density, MTT (250mg/ml) was added to the cellsand incubated for 2 h at 37°C. Subsequently the medium was carefullyremoved in such a way that any detached or loosely adherent cells were notremoved, and the cells containing the trapped MTT crystals were solubilizedin 200 ml DMSO at 37°C for 15 min. Absorbance was determined in amicrotiter plate reader (Molecular Devices, Menlo Park, CA) at 550 nm andsubtracted from theA650 nm.

Cell Adhesion. Cell adhesion was assayed by plating 100,000 trypsinizedcells onto wells of a 48-well non-tissue culture-treated plate coated overnightwith vitronectin (2 mg/ml) or the 110-kDa RGD-containing fragment offibronectin (10mg/ml), which were washed and blocked with 0.5% BSA inPBS. After 1 h, the wells were washed 3 times to remove nonadherent cells,and the remaining cells were quantitated using the MTT assay as describedabove.

Hoechst Stain. BBEC were stained with Hoechst-bisbenzamide 33258(Sigma) according to the manufacturer’s instructions to assess chromatincondensation and nuclear fragmentation. Approximately 300 cells in eachsample were counted under UV filter at magnification3400.

JNK Activation. Lysates from 53 106 BBEC that have been exposed toC2-ceramide were prepared in Triton X-100 extraction buffer [EB buffer: 1%Triton X-100, 10 mM Tris (pH 7.6), 50 mM NaCl, 0.1% BSA, 1 mM PMSF, 1%aprotinin, 5 mM EDTA, 50 mM NaF, 0.1% 2-b mercaptoethanol, 5mM

phenylarsine oxide, and 100mM vanadate], precleared (15,0003 g for 45 minat 4°C), and anti-SAPK/JNK immunoprecipitation was performed using anti-SAPK1/JNK1 rabbit polyclonal antibodies (Santa Cruz Biotechnology, SantaCruz, CA) as previously described (46). Immunoprecipitates were washedseveral times with buffer [20 mM PIPES (pH 7.0), 0.1 M NaCl, and 20mg/mlaprotinin], and kinase activity of JNK was quantitated using a slight modifi-cation of anin vitro kinase assay as described (47). JNK immunoprecipitateswere incubated in a solution containing 20 mM PIPES (pH 7.0), 10 mM MnCl2,5 mCi g[32P]ATP (3000 Ci/mmol), 20mg/ml aprotinin, and 1mg of GST-c-junfusion protein (NH2 terminus, residues 1–154) in final volume of 20ml. Afteran incubation of 30 min at 30°C, the reactions were terminated by the addition

of 20 ml of 23 SDS-sample buffer and heated at 98°C for 5 min. The JNKkinase and GST-c-jun substrates were resolved by SDS-PAGE, phosphorylatedGST-jun protein was visualized by autoradiography, and the amount of JNKkinase protein immunoprecipitated was verified by anti-JNK Western blotting.

Statistical Analyses.All laboratory and histopathological analyses wereperformed independently and without knowledge of clinical data. Immunohis-tochemical slides prepared from serial sections were analyzed by two observ-ers (A. E-E. and H. S.), who visually determined the proportion of microves-sels in the whole section in which the microvascular endothelium specificallystained with the LM609 or P1F6 mAb compared with the total Factor VIII-positive vessels in the adjacent section. The observers repeated the analysis ofthe sections on two separate occasions and were blinded to the clinicalcharacteristics of the patients and to their previous readings. The variation forintegrinavb3 expression between the first and second reading in the 40 tumorsexamined was 15% in 2 tumors, 10% in 9 tumors, and 0–5% in the remaining29 tumors. The variation for integrinavb5 between the first and secondreadings in the 21 tumors evaluated was 15% in 1 tumor, 10% in 6 tumors, and0–5% in 13 tumors. The value used for statistical analysis and for plotting thefigures was the average of the readings for each tumor. A simple linearregression analysis was used to evaluate the association of age (as a continuousvariable and also dichotomized as#12 monthsversus.12 months), MKI(low 1 intermediateversushigh; Ref. 3), pathology (favorableversusunfa-vorable), MYCN (not amplifiedversusamplified), and whether or not thepatient had received treatment prior to tumor sampling, with expression ofaVb3 andaVb5 as measured by the percent of microvessels that stained withthe antibodies LM609 (foraVb3) and P1F6 (foraVb5). TheP values reportedare based on theF test from the regression analysis and are all two-sided(Table 1). Means and 95% confidence intervals based on the individual SDwere calculated. To summarize the association between expression ofaVb3

andaVb5, scatter plots were drawn and Pearson’s correlation coefficient wascalculated with its associatedP value (Fig. 4). To test whether the observedcorrelation could be explained by MYCN amplification, a partial correlationwas calculated. To evaluate whether the association betweenaVb3 andaVb5

was similar or different in patients whose tumors expressed MYCN amplifi-cation or not, separate regression models were fit for the two subgroups ofpatients and were compared with the single model fit for all 21 patients.

Table 1 Most microvessels in high-risk NB express integrinavb3

No. ofpatients (%)

Mean % of microvesselsexpressingavb3

(95% confidence interval)UnivariateP for association with% microvessels expressingavb3

StageI 5 (13) 14% (5–23) ,0.001a

II 5 (13) 22% (9–35)III 5 (13) 60% (15–100)IV 25 (62) 58% (51–66)

Age#1 year 16 (40) 40% (28–53) 0.12.1 year 24 (60) 54% (42–65) (0.12 when age was continuous)

MKILow 17 (44) 37% (24–51) 0.004Intermediate 7 (18) 45% (24–65)High 15 (38) 65% (56–75)

PathologyFavorable 20 (50) 33% (22–43) ,0.001Unfavorable 20 (50) 64% (56–73)

MYCNNot amplified 23 (58) 36% (25–46) ,0.001Amplified 17 (42) 66% (57–74)

Stage and MYCNI/II and III-not amplified 12 (30) 18% (13–24) ,0.001III-amplified & IV 28 (70) 61% (54–69)

Treatment prior to sampling in high risk:stage III, MYCN-amplified and stage IV

Previously untreated 23 (82) 59% (50–67) 0.17Previously treated 5 (18) 72% (56–88)

Stage and MYCNI/II-not amplified 10 (25) 18% (11–25) Comparing not-MYCN-amplified to MYCN-amplified:

P , 0.001 (stage III) andP 5 0.36 (stage IV)III-not amplified 2 (5) 20% (20–20)III-amplified 3 (8) 87% (79–94)IV-not amplified 11 (28) 54% (40–69)IV-amplified 14 (35) 61% (53–70)

a All P are based onF test for effect in regression model with only one baseline characteristic (the degrees of freedom in the numerator depends on the number of levels).

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RESULTS

Integrin avb3 Is Expressed on Angiogenic Endothelial Cells ofHigh-Risk NB. To determine whether NBs could potentially respondto anti-integrin avb3 treatment, we examined whether the tumorvasculature expressed this integrin. Serial sections from 40 NB tumorswere immunohistochemically stained with anti-Factor VIII or theanti-integrinavb3 (LM609) antibodies and analyzed for microvascu-lar expression of integrinavb3. Fig. 1 demonstrates representativefields from serial sections stained with mAb LM609 and anti-FactorVIII from two patients with a high-risk or a low-risk NB. Anti-FactorVIII stains endothelial cells in microvessels of both tumors (Fig. 1,CandD). A large proportion of the microvessels which were positivefor Factor VIII in the high-risk case (Fig. 1C) also stained positivelywith mAb LM609 (Fig. 1A), indicating endothelial expression ofintegrinavb3. In the low-risk NB, there was no endothelial expressionof integrin avb3 (Fig. 1B). Also of note is the low cellularity of thelow-risk, non-MYCN-amplified NB (Fig. 1,B andD) compared withthe high cellularity of the high-risk tumor with the high MYCN copynumber (Fig. 1,A andC). Additional serial sections from each casewere stained with isotype-specific mouse IgG (IgG1, negative control)to verify specificity of the LM609 stain, and with standard H&E tomonitor the quality of the OCT frozen sections (data not shown).Staining of NB tumor samples with a mAb specific to the integrinav

chain (LM142) confirmed an endothelial expression similar to that ofintegrin avb3 (data not shown).

Endothelial localization of integrinavb3 was further demonstratedby simultaneous immunostaining of the same section for both integrinavb3 and Factor VIII using a mAb to integrinavb3 (LM609) and arabbit polyclonal antibody to Factor VIII (Fig. 2) as described (12).

The immunofluorescent stain demonstrates that the microvascularendothelium of this tumor expresses integrinavb3 (red fluorescence,mAb LM609, Fig. 2B) and Factor VIII (green fluorescence, poly-clonal anti-Factor VIII antibody, Fig. 2A) but that the surroundingtumor cells do not express them. The colocalization of integrinavb3

to the endothelium was further verified by double exposure of thesame frame to the green and red fluorescence, resulting in a yellowfluorescence in the regions where both Factor VIII and integrinavb3

were colocalized (Fig. 2C). Supporting these observations, the immu-nohistochemical staining for integrinavb3 was limited to the endo-thelial cells in all cases examined and was not detected on the tumorcells themselves (Fig. 1,A andC).

Fig. 3 and Table 1 summarize the findings in the 40 NB tumorsstained as described in Fig. 1 and demonstrate that integrinavb3 ishighly expressed on most microvessels in high-risk but not low-riskNB. Univariate analysis demonstrates that the high-risk tumors (stageIII, MYCN-amplified, and stage IV) expressed endothelial integrinavb3 on the majority of their microvessels (mean, 61%; 95% confi-dence interval, 54–69%;n 5 28; Table 1 and Fig. 3). The highproportion of microvessels expressing the integrinavb3 in high-riskNBs was found both in tumor specimens obtained at the time ofdiagnosis (mean, 59%; 95% confidence interval, 50–67%;n 5 23)and in tumors procured at the time of delayed resection or tumorrelapse (mean, 72%; 95% confidence interval, 56–88%;n 5 5). Incomparison, a significantly lower proportion of microvessels ex-pressed endothelial integrinavb3 in low-risk tumors (stages I and IIand stage III non-MYCN-amplified; mean, 18%; 95% confidenceinterval, 13–24%;n 5 12; Fig. 3 and Table 1). Notably, in the fivestage III tumors, there was a striking difference in expression of

Fig. 1. Microvessels of high-risk NB express integrinavb3. Frozen sections of Stage III NBs [Aand C (high-risk): MYCN-amplified, unfavorable Shimada classifica-tion, and high MKI, resected from a patient diagnosed atage 47 months;B and D (low-risk): non-MYCN-ampli-fied, favorable histology by Shimada classification, andlow MKI, resected from a patient diagnosed at age 33months] were stained by immunoperoxidase for integrinavb3 (A andB, mAb LM609) and Factor VIII (CandD)as described in “Materials and Methods.”Brown colorrepresents positively stained microvessels. Percent posi-tivity of microvessels for integrinavb3 and avb5 wasdetermined as a fraction of the vessels which stained forFactor VIII in consecutive serial sections. A representativefield is shown. Photographed at magnification3100.

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integrin avb3 between the MYCN-amplified/unfavorable histologyand MYCN non-amplified/favorable histology tumors (mean, 87%andn 5 3 versusmean, 20% andn 5 2, respectively; Figs. 1 and 3and Table 1). In a multiple-regression analysis with stage and MYCNin the model, stage and MYCN were very strong predictors of integrinavb3 expression (i.e.,LM609 staining) on the tumor neovasculature(P , 0.001), whereas MKI and pathology were not (P 5 0.15 foreach). We conclude that integrinavb3 is highly expressed on mi-crovessels of high-risk NBs.

Association between Expression of Integrinavb3 and avb5 inHigh-Risk Neuroblastomas.Another av integrin, avb5, is also ex-pressed on endothelium of angiogenic vessels and, like integrinavb3,is crucial to angiogenesis (8). We asked whether integrinavb5 also isexpressed in angiogenic endothelium in NBs. Twenty-one of the 40NBs (all stage IV) were evaluated for expression of integrinavb5

(Fig. 4). Sixteen of the 21 expressed integrinavb5 on 50% or more oftheir angiogenic vessels (Fig. 4). Mean expression of integrinavb5

was 60% (95% confidence interval, 53–67%;n 5 21). The correlationbetween expression of integrinavb3 and avb5 was high (r5 0.75;P , 0.001). When controlling (i.e., stratifying) for MYCN status, thepartial correlation coefficient becamerp 5 0.74 (P, 0.001), suggest-ing that both integrins play a role in tumor angiogenesis independentof MYCN amplification. Like integrinavb3, integrinavb5 was onlyexpressed on the angiogenic endothelium and not by tumor cells inany of the cases we examined (data not shown). These data demon-strate that the angiogenic integrinsavb3 andavb5 are strongly asso-ciated and are expressed on most angiogenic microvessels in high-risk

Fig. 3. Integrinavb3 is expressed by microvessels of high-risk but not low-risk NBs.Serial frozen sections of 40 cases of NB of different stages and MYCN amplification, assummarized in Table 1, were stained for integrinavb3 (mAb LM609) and Factor VIII (seeFig. 1), and the fraction of the vessels expressing integrinavb3 (expressed as a percentageof the vessels expressing integrinavb3) was determined as detailed in “Materials andMethods.”M, stage I non-MYCN-amplified tumors;‚, stage II non-MYCN-amplifiedtumors;L, stage III non-MYCN-amplified tumors;l, stage III MYCN-amplified tu-mors;E, stage IV nonamplified tumors;F, stage IV MYCN-amplified tumors.

Fig. 2. Integrinavb3 expression is localized to endothelial cells inNBs.A single cryostat section from a MYCN-amplified stage IV NBwith unfavorable Shimada classification resected from a child diag-nosed at age 8 months was dual-stained with both mAb LM609directed to integrinavb3 and polyclonal rabbit anti-Factor VIIIantibody. After incubation with the primary antibodies, the samplewas reacted with two secondary antibodies, Rhodamine-conjugatedanti-mouse IgG and FITC-conjugated anti-rabbit IgG, as describedin “Materials and Methods.” Immunofluorescence was detected withan Olympus AX70 compound microscope at magnification3200.Green indicates Factor VIII-expressing endothelium (A),red indi-cates expression of integrinavb3 (stained with mAb LM609;B), andyellow demonstrates colocalization of integrinavb3 to the FactorVIII-positive endothelial cells (achieved by using double exposureof the film, first using the green fluorescence and then the redfluorescence, resulting in a yellow fluorescence where they weresuperimposed;C).

Fig. 4. Expression of endothelial integrinavb3 correlates with expression of integrinavb5 in stage IV NB. Expression of integrinsavb3 andavb5 on tumor microvessels wasdetermined in frozen sections of 21 cases of stage IV NB (F, MYCN-amplified;E,non-MYCN-amplified) as described in “Materials and Methods.” The results are ex-pressed as the percentage of Factor VIII-positive microvessels that stain with LM609(avb3) plotted against the percentage of Factor VIII-positive microvessels that stain withP1F6 (avb5) in each of the tumors analyzed. The calculated Pearson’s correlationcoefficient between microvascular endothelial expression ofaVb3 and aVb5 in the 21tumors analyzed wasr 5 0.75 (P, 0.001).

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NBs. This suggests that both integrins may present a useful target forantiangiogenic treatment of high-risk NB.

Inhibition of Integrin-mediated Endothelial Cell Anchorage IsAssociated with Elevation of Endogenous Ceramide.Integrin-mediated signals are required for anchorage-dependent survival ofendothelial and epithelial cells (21, 48, 49). Inhibition of integrinsavb3 and avb5 on endothelial cells lining tumor neovasculature inanimal models induces endothelial cell apoptosis, disruption of tumorangiogenesis, and inhibition of tumor growth (8, 11, 14). Endothelialcells also require integrin-mediated attachment and spreading onspecific matrix proteins to survive in culture (14, 21, 48). Because weshowed that endothelium in high-risk NBs expressed integrinsavb3

andavb5 (Figs. 1–4), we sought to further study the signaling path-ways that may contribute to endothelial apoptosis mediated via inhi-bition of these integrins.

On the basis ofin vitro and in vivo experiments demonstratinginvolvement of ceramide in UV irradiation, LPS, and TNFa-inducedendothelial apoptosis (17, 19, 23), we hypothesized that ceramidecould be involved in endothelial cell death resulting from inhibition ofintegrin-mediated anchorage. Because NB is a neural crest-derivedtumor, in testing this hypothesis we chose to use BBEC immortalized(large-T transfected) endothelial cells derived from a neural microen-vironment (the brain). Like other endothelial cells, BBEC spontane-ously express high levels of integrinavb3 on their surface. Cellularlipids of attached, log-phase growth BBEC were labeled with[3H]palmitic acid, detached briefly by trypsin/EDTA, and then re-plated on BSA, fibronectin, or vitronectin for 4 h. Lipids wereextracted and separated by TLC as detailed in “Materials and Meth-ods.” Prevention of attachment and spreading of endothelial cells ontoextracellular matrix by plating them on a BSA-coated surface inducedelevation of ceramide of up to 50% above that observed in cells platedon fibronectin or vitronectin (Fig. 5A). The mean increase in endog-enous ceramide in BBEC plated on BSA was 35% (SD, 15%;n 5 5experiments) compared with vitronectin and 29% (SD, 9%;n 5 3experiments) compared with fibronectin. These results were replicatedin primary HUV-EC-C. An increase of 54% in endogenous ceramidewas observed in the HUV-EC-C when they were plated on BSAcompared with vitronectin (SD, 1.336 0.1% [3H]ceramide on BSAcompared with 0.876 0.01% on vitronectin).

Blockade of integrinsavb3 andavb5 using function-blocking an-tibodies or the cyclic pentapeptide RGDfV, which block the RGD-binding site on these integrins, has been reported to prevent endothe-lial cell attachment to matrix and to induce apoptosis (5, 8, 11, 12).Exposure of BBEC to the RGD-blocking cyclic pentapeptide RGDfVbut not to the control peptide RADfV prevented BBEC attachmentand spreading on vitronectin in a dose-dependent pattern, with com-plete inhibition achieved with as low as 5mg/ml RGDfV (Fig. 5Banddata not shown). Similar inhibition of attachment and spreading byRGDfV was observed on surfaces coated with the RGD-containing110-kDa fragment of fibronectin (data not shown). BBEC that wereprevented from attaching to vitronectin by RGDfV (10mg/ml) dem-onstrated chromatin condensation and nuclear fragmentation by Ho-echst stain in 37.56 5% of the cells, indicating that inhibition of cellanchorage mediated by RGD engagement of vitronectin-binding in-tegrins in BBEC induces apoptosis (Fig. 5C). In contrast, only8.76 3% of cells incubated with the control peptide RADfV (Fig. 5C)exhibited these apoptotic features, a result similar to that of cellsplated on vitronectin without inhibitory peptide (6.66 3%; Fig. 5C).The specific inhibition of integrin-dependent attachment/spreadingonto vitronectin by the RGDfV cyclic peptide was associated with aconcomitant increase in endogenous ceramide levels (Fig. 5D). InBBEC plated in the presence of RGDfV, endogenous [3H]ceramideincreased by up to 50% above the levels found in the presence of the

control peptide RADfV. This increase in endogenous ceramide wassimilar to the increase observed when the BBEC were plated on aBSA-coated surface (Fig. 5D). Ceramide content in cells treated withthe control peptide RADfV was similar to that of control cells adher-ing and spreading on vitronectin in the absence of added peptide (Fig.5D). The increase in endogenous ceramide was observed at peptideconcentrations similar to those that inhibited BBEC spreading andattachment onto vitronectin and increased apoptosis (10–500mg/ml).

Fig. 5. Apoptosis induced by inhibition of integrin-mediated anchorage on vitronectinis associated with increased endogenous levels of ceramide in endothelial cells.A, BBEC(106 cells/sample) labeled with 1mCi/ml 9,10 [3H]-(N)-palmitic acid were plated onnon-tissue culture-treated 6-well plates coated with BSA (0.5%), vitronectin (2mg/ml), orfibronectin (10 mg/ml). After 4 h, lipids were extracted and ceramide content wasanalyzed by TLC. TNFa (500 ng/ml) was added to one set of triplicate samples plated onvitronectin 30 min before extraction of lipids (positive control). Ceramide is expressed asthe percentage of total [3H]-labeled lipids extracted.Bars represent mean of the percentceramide extracted from cells from three identical samples plated on BSA, vitronectin, orfibronectin, or treated with TNFa in this experiment.Error bars represent SD. The resultsrepresent a typical experiment, repeated 10 times.B, BBEC (105 cells per well) wereplated in a vitronectin-coated non-tissue culture-treated 48-well plate with concentrationsof RGDfV (F) or RADfV (E) between 0 and 50mg/ml and were allowed to adhere andspread for 60 min. The wells were then washed three times to remove nonadherent cells,and the remaining adherent cells were quantitated by MTT assay (see “Materials andMethods”).Data pointsanderror barsare means and SD of six replicate samples; in datapoints where theerror bar is smaller than the symbol, it is hidden by it.C, BBEC (105)were plated in a 48-well non-tissue culture-treated plate coated with vitronectin (2mg/ml)in the presence of 10mg/ml cyclic RGDfV (f), RADfV (control;M), or without addition(control;o) and were incubated for 6 h. Cells were trypsinized, and a cytospin from eachsample was stained with Hoechst-bisbenzamide to demonstrate nuclear fragmentation andcondensation.Bars represent mean percentage of condensed nuclei per high power fieldof total nuclei in the field (magnification3400) counted in five different fields of eachcytospin.Error bars represent the SD of the count of the five high power fields.D, Thecyclic RGD-blocking peptide RGDfV [10mg/ml (o) or 0.5 mg/ml (f)] or control peptideRADfV [0.5 mg/ml (M)] were added to 106 [ 3H]palmitic acid-labeled BBEC and thenplated on vitronectin as inA. For comparison, cells were also plated on BSA [positivecontrol (f)] or vitronectin [negative control (M)] without additions. After 4 h, lipids wereextracted and ceramide was quantitated as above. Data are presented as mean6 SD(n 5 3) of the percent ceramide extracted from three identical samples of cells treatedunder the same conditions;error bars represent the SD of the triplicate samples.

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These data suggest that endogenous ceramide participates in signalingof apoptosis, induced by inhibition of integrin-mediated endothelialcell anchorage.

Ceramide-induced Cytotoxicity Is Associated with the Activa-tion of JNK. Some stress signals that lead to apoptosis in endothelialcells induce endogenous ceramide, which is associated with activationof JNK, a MAPK that functions in apoptosis (23). We showed thatinhibition of integrin-mediated endothelial cell anchorage, which re-sults in apoptosis, induces elevation of endogenous ceramide. Weasked whether ceramide itself could induce JNK activation and BBECdeath. C2-ceramide was cytotoxic to BBEC with a mean concentrationwhich induces 50% cell death (LC50) of 31.7 mM (SD, 10.5mM) at24 h, as determined in eight separate experiments (each done inreplicates of 5–10 points for each ceramide concentration). The deathresponse was proportional to the concentration of ceramide added(Fig. 6A) and to the length of time BBEC were exposed to the lipid(Fig. 6B). The inactive ceramide analogue dihydroceramide elicitedonly a limited amount of cell death, which was constant and not dosedependent between 20 and 100mM dihydroceramide (data not shown).Exposure of BBEC to increasing concentrations (0.1–40mM) ofexogenous C2-ceramide for 2 h induced activation of JNK, as assayedby measurement of phosphorylation of GST-c-jun fusion protein byJNK immunoprecipitated from the cells (Fig. 7). The increase in JNKphosphorylation of the GST-c-jun fusion protein was observed asearly as 1 h after the addition of C2-ceramide, with the largestincrement in the increase in JNK activation occurring between 1 and2 h or later (data not shown). Thus, exogenous C2-ceramide, whichinduces apoptotic cell death (45), also induces the activation of JNKin endothelial cells. From these data we conclude that ceramide is apotential participant in detachment-induced apoptotic signaling inendothelial cells and, specifically, that it may mediate some of thesignals associated with integrin blockade.

DISCUSSION

Our data constitute the first report of expression of integrinsavb3

and avb5 by microvascular endothelium of a pediatric tumor andassociation of this expression with tumor aggressiveness. Becausethese integrins are markers of active angiogenesis (8, 14), this findingextends the data of Meitaret al. (30), who reported that a high

vascular index in NB correlates with metastatic disease, MYCNamplification, and poor outcome. In invasive breast cancer, endothe-lial integrin avb3 expression and microvessel density are both prog-nostic indicators (50, 51), suggesting that this correlation also may befound in other tumors. Our ongoing analysis of NBs will revealwhether endothelial integrinavb3 expression is a prognostic factorindependent from MYCN and pathology in NB. Demonstration thatangiogenic endothelium in high-risk NBs highly expressed integrinsavb3 and avb5 suggests that these integrins could be therapeutictargets.

Our data showing that microvessels in MYCN-amplified NB ex-press high levels of integrinsavb3 andavb5 point to a potential rolefor the MYCN oncogene in angiogenesis. Because MYCN itself is atranscription factor, it could regulate angiogenic factors originating inthe tumor cells, such as vascular endothelial growth factor or matrix-degrading proteins, as has been shown for mutant K-ras and polyomamiddle T antigen, respectively (52, 53). Supporting this, NB tumorsand their surrounding stromal cells express matrix metalloproteinases(31). Moreover, MYCN is required for motility of NB cell lines andfor their proteolytic activity in vitro, suggesting that MYCN maycontribute to NB aggressiveness by enhancing angiogenesis (54).Thus, it is possible that overexpression of MYCN may contribute toNB aggressiveness by promoting angiogenesis.

The immunohistochemical stains of the NBs in our series showedthat integrinavb3 andavb5 expression was confined to the endothe-lium of tumor microvessels and did not appear on the tumor cellsthemselves (Figs. 1 and 2 and data not shown). Gladsonet al. (55)examined paraffin sections of NBs for expression of the individualav,b3, and b5 integrin subunits. Our immunostaining with the mAbagainstavb5 (P1F6) are in agreement with their description that theb5

integrin subunit is not expressed at the protein level in neuroblasts ofundifferentiated tumors. However, Gladsonet al. (55) observed thatthe av andb3 subunits were associated with most of the undifferen-tiated neuroblasts, a finding interpreted to imply that integrinavb3 isexpressed on those cells. It is conceivable that the binding partner(s)for the av andb3 integrin chains seen in that work did not belong tointegrinavb3 but may have originated from different integrin partners(i.e.,avb6 or avb8; Refs. 56–58), some of which are highly expressedin the mammalian brain (58). This could explain the specific micro-

Fig. 7. C2-ceramide induces activation of JNK in endothelial cells. BBEC (53 106

cells/sample) were exposed to C2-ceramide (0–40mM) for 2 h, harvested, lysed, andimmunoprecipitated witha-JNK antibody. Kinase activity of JNK was quantitated usingGST-C-jun fusion protein as substrate, which was resolved on 10% SDS-PAGE. Thisautoradiograph demonstrates phosphorylation of the GST-C-jun fusion protein substrateby the JNK immunoprecipitated from C2-ceramide-treated BBEC lysates. Positive controlwas obtained by measuring JNK activation in BBEC (53 106) exposed to UV radiation(25 mjoules) and then incubating for 2 h before assessment of JNK activity as describedabove. Equal amounts of precipitated JNK were verified bya-JNK immunoblots (data notshown).

Fig. 6. Exogenous C2-ceramide is cytotoxic to BBEC.A, BBEC (2 3 104 cells/well)were incubated in a 96-well plate with 0–80mM C2-ceramide for 24 h. Cell viability wasassessed by MTT assay at 24 h as detailed in “Materials and Methods.” Eachdata pointrepresents the mean of six replicate wells;error bars represent SD of the six replicates,and where they are smaller than the symbols, they are hidden by them.B, Details are asin A, except that BBEC were incubated with 30mM (F) or 50 mM C2-ceramide (E) fordifferent lengths of time, washed and incubated without ceramide.

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vascular endothelial expression of integrinavb3 we found using mAbLM609, which specifically recognizes the native conformation of thefunctional RGD-binding domain on integrinavb3 (7, 59).

We demonstrate here for the first time that ceramide is elevated inendothelial cells that are prevented from attaching and spreading onmatrix, either by plating on BSA or by inhibition with the RGDfVinhibitory peptide (Fig. 5). The increase in endogenous ceramideinduced by RGDfV and plating on BSA are in a range comparablewith increases observed following exposure to stressors such asTNFa, sorbitol, H2O2, LPS, UV irradiation, org irradiation (17, 60).Our data suggest that ceramide may be involved in signaling pathwaysoriginating from specific inhibition of the RGD binding site onvitronectin-binding integrins such asavb3 and avb5. Although wecannot exclude possible involvement of multiple vitronectin-bindingintegrins under these conditions, it is likely that inhibition of adhesionand spreading on vitronectin by RGDfV was mediated mostly byinhibition of integrins avb3 and avb5, both of which are highlyexpressed on endothelial cells (7, 9). Alternatively, increased ceram-ide may be the result of inhibition of cell anchorage and/or of theassociated cytoskeletal signals resulting from shape changeper se(61), may be unrelated to the alteration of integrin signaling, orpossibly may be related to direct activation of apoptosis by an RGD-blocking peptide (62). To implicate specific signaling intermediatesthat may link integrins, ceramide, and regulation of cell survival (i.e.,phosphatases such as protein tyrosine phosphatase-a or kinases suchas AKT/PKB) and to determine the role of cytoskeletal shape changeversus integrin blockade in cell survival, it will be necessary toexplore these pathways in detail by using specific dominant-negative/positive intermediates or anti-integrin antibodies that block signalingwithout inhibiting cell attachment (63). Although there have been noprior reports of ceramide as a second messenger in integrin signaling,recent work suggests that selectins, another family of cell adhesionmolecules, may use ceramide signaling in the activation of lympho-cytes (64). Increased endogenous ceramide may contribute to endo-thelial apoptosis, as shown by the fact that incubation of endothelialcells with C2-ceramide is associated with DNA laddering (data notshown and Ref. 45). Our results compare favorably with those de-scribed by Xuet al. (45), as can be seen in the fact that we achieveda mean LC50 of 31.7mM 6 10.5 SD C2-ceramide in our experimentscompared with their report of 50% endothelial cell kill at 50mM

C2-ceramide. Our data provide the first evidence that signaling path-ways originating from inhibition of endothelial cell anchorage areassociated with increased endogenous ceramide, which may contrib-ute to apoptosis.

In several cell types including endothelial cells, JNK is thought tolie downstream of ceramide in apoptotic pathways induced by stress(23, 65). In epithelial cells, detachment from matrix induces JNKactivation, which can be inhibited by the interleukin 1b convertingenzyme protease inhibitor crmA or by overexpression of Bcl-2 (66).Our experiments, in which exogenous C2-ceramide induced activationof JNK in endothelial cells, suggest that the increased endogenousceramide we observed after inhibition of integrin-mediated cell an-chorage (Fig. 5) may contribute to JNK activation in signaling ofapoptosis (Fig. 7). However, the ceramide-JNK-apoptosis link is notuniversal, and several reports describe the dissociation of ceramideproduction from JNK and other death signals (67–70). Recent work byAmeyar et al. (67) described lack of correlation between JNK acti-vation and ceramide production in breast carcinoma cells, suggestingthat the ceramide-JNK link may be specific to individual cell types.Adding to the complexity, Khwaja and Downward (68) show that,although JNK is activated by detachment of normal MDCK epithelialcells, it is unlikely that it plays a direct role in detachment-inducedapoptosis in these cells because activated phosphatidylinositol 39-

kinase and AKT prevented apoptosis but did not prevent the associ-ated JNK activation. Activated Raf and dominant negative SAPK/ERK kinase-1, which inhibited the detachment-induced activation ofJNK, did not prevent apoptosis, suggesting induction of apoptosis byelements which are independent of JNK activation in the MDCKepithelial cells (68). Clearly the specificity of cellular responses toceramide and other apoptosis-mediating signals depends upon manyfactors including the nature of the stimulus, costimulatory signals, thecell type involved, and (most likely) the surrounding matrix proteins.Thus, although inhibition of endothelial cell anchorage induced ele-vation of ceramide in our studies and exogenous ceramide inducedJNK activation and apoptotic cell death, a direct causal link betweenthe three needs to be investigated further.

Cell survival is dictated by a delicate dynamic balance between pro-and anti-apoptotic signals, which in turn depend upon a complexinterplay of factors such as integrins and growth factors. For example,in fibroblasts interleukin-1-mediated inflammatory responses, whichinduce activation of the proapoptotic JNK/SAPK, and the survival-mediating NFkB are both modulated by integrin binding to RGDmotifs on fibronectin (71). Both the JNK/SAPK and NFkB pathways,as well as phosphatidylinositol 39-kinase/AKT, are tightly controlled,and several reports suggest that ceramide may help regulate them totip the balance in favor of apoptosis (60, 72–75). These reportssupport our finding that ceramide may be involved in regulation ofanchorage-induced signaling in endothelial cells. Furthermore, NFkBmediates integrinavb3-dependent endothelial cell survival (16),whereas ceramide levels increase in endothelial cells upon loss ofintegrin-dependent matrix anchorage (Fig. 5). It is possible that underconditions of inhibition of cell anchorage ceramide mediates signalsto down-regulate NFkB activation in the endothelial cells, thus pro-moting their apoptosis through activation of JNK/SAPK (Fig. 7 andRefs. 23, 66, and 72). It is also possible, that stress conditions whichaugment ceramide and/or JNK may synergistically increase apoptosisassociated with loss of anchorage-specific survival signals, therebyincreasing any antiangiogenic activity of the RGDfV peptide.

The relationship between ceramide generation and caspase activa-tion is dependent on cell type and the apoptotic stimulus used. Atpresent, the relationship between ceramide metabolism and regulationof caspase activation in our system has been only preliminarilydefined. Both nuclear fragmentation of BBEC plated on vitronectin inthe presence of RGDfV and DNA laddering in BBEC plated onBSA-coated plates are inhibited by the pan-caspase inhibitor, BOC-D-FMK (data not shown). In preliminary experiments BOC-D-FMKdid not prevent the increase in endogenous ceramide in BBEC platedon vitronectin for 4 h in the presence of RGDfV, suggesting thatceramide may be upstream of the BOC-D-FMK-inhibitable caspases(data not shown). Consistent with this, initial experiments show thatDNA laddering induced by exogenous C2-ceramide can be inhibitedby BOC-D-FMK (data not shown). This suggests a situation similar tothat found in apoptosis induced by low-dose okadaic acid in humanneuroepithelioma cells in which endogenous ceramide accumulationis insensitive to caspase inhibitors and precedes caspase activation,whereas exogenous C6-ceramide-induced apoptosis was inhibited bythe caspase inhibitors (76, 77). In other types of apoptotic signals,such as those mediated by TNF superfamily receptors, the initiatorcaspases are thought to be upstream of ceramide generation, whereaseffector caspases are downstream of it (78–81). Because specificsignaling cascades may differ depending on the stimulus and the celltype (78), this needs to be examined individually for each cell typeand experimental model. Ongoing investigations using BBEC as amodel seek to determine the direct relationship between ceramide andendothelial anoikis.

In summary, this is the first description that suggests that integrins719

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avb3 and avb5 are highly expressed on the microvascular endothe-lium in a pediatric malignant tumor, high-risk neuroblastoma. Alsonovel is our finding that endothelial cell apoptosis, induced by inhi-bition of vitronectin-binding integrins and the prevention of endothe-lial cell anchorage on matrix, is associated with increased endogenousceramide. Taken together, these data suggest that RGD-dependent,vitronectin-binding integrins on endothelial cells, such asavb3 andavb5, may be potential targets for antiangiogenic treatment of NB.

ACKNOWLEDGMENTS

We thank Pei-Gen Wu, Yenbou Liu, and Dennis Duncan for their excellenttechnical assistance. We also thank Drs. C. P. Reynolds, B. Maurer, and N.Keshelava for their expert advice and assistance with the ceramide assays.Special thanks to Dr. D. A. Cheresh for generously supplying mAb LM609 andto Dr. A. Jonczyk (Merck, Darmstadt, Germany) for the generous gift of theRGDfV and RADfV cyclic peptides. Our thanks also to the donation frommembers of Riviera Hall Lutheran School in Los Angeles in support of thisresearch.

REFERENCES

1. Felding-Habermann, B., and Cheresh, D. A. Vitronectin and its receptors. Curr. Opin.Cell Biol., 5: 864–868, 1993.

2. Huang, X., Wu, J., Spong, S., and Sheppard, D. The integrinavb6 is critical forkeratinocyte migration on both its known ligand, fibronectin, and on vitronectin.J. Cell Sci.,111: 2189–2195, 1998.

3. Nishimura, S. L., Sheppard, D., and Pytela, R. Integrinavb8: interaction withvitronectin and functional divergence of theb8 cytoplasmic domain. J. Biol. Chem.,269: 28708–28715, 1994.

4. Schnapp, L. M., Hatch, N., Ramos, D. M., Klimanskaya, I. V., Sheppard, D., andPytela, R. The human integrina8b1 functions as a receptor for tenascin, fibronectin,and vitronectin. J. Biol. Chem.,270: 23196–23202, 1995.

5. Pfaff, M., Tangemann, K., Muller, B., Gurrath, M., Muller, G., Kessler, H., Timpl, R.,and Engel, J. Selective recognition of cyclic RGD peptides of NMR defined confor-mation byaIIbb3, aVb3, anda5b1 integrins. J. Biol. Chem.,269:20233–20238, 1994.

6. Aumailley, M., Gurrath, M., Muller, G., Calvete, J., Timpl, R., and Kessler, H.Arg-Gly-Asp constrained within cyclic pentapeptides: strong and selective inhibitorsof cell adhesion to vitronectin and laminin fragment P1. FEBS Lett.,291: 50–54,1991.

7. Cheresh, D. A. Human endothelial cells synthesize and express an Arg-Gly-Asp-directed adhesion receptor involved in attachment to fibrinogen and von Willebrandfactor. Proc. Natl. Acad. Sci. USA,84: 6471–6475, 1987.

8. Friedlander, M., Brooks, P. C., Shaffer, R. W., Kincaid, C. M., Varner, J. A., andCheresh, D. A. Definition of two angiogenic pathways by distinctav integrins.Science (Washington DC),270: 1500–1502, 1995.

9. Klein, S., Giancotti, F. G., Presta, M., Albelda, S. M., Buck, C. A., and Rifkin, D. B.Basic fibroblast growth factor modulates integrin expression in microvascular endo-thelial cells. Mol. Biol. Cell,4: 973–982, 1993.

10. Christofidou-Solomidou, M., Bridges, M., Murphy, G. F., Albelda, S. M., andDeLisser, H. M. Expression and function of endothelial cellav integrin receptors inwound-induced human angiogenesis in human skin/SCID mice chimeras. Am. J.Pathol.,151: 975–983, 1997.

11. Brooks, P. C., Montgomery, A. M., Rosenfeld, M., Reisfeld, R. A., Hu, T., Klier, G.,and Cheresh, D. A. Integrinavb3 antagonists promote tumor regression by inducingapoptosis of angiogenic blood vessels. Cell,79: 1157–1164, 1994.

12. Brooks, P. C., Stromblad, S., Klemke, R., Visscher, D., Sarkar, F. H., and Cheresh,D. A. Anti-integrin avb3 blocks human breast cancer growth and angiogenesis inhuman skin. J. Clin. Invest.,96: 1815–1822, 1995.

13. Friedlander, M., Theesfeld, C. L., Sugita, M., Fruttiger, M., Thomas, M. A., Chang,S., and Cheresh, D. A. Involvement of integrinsavb3 andavb5 in ocular neovasculardiseases. Proc. Natl. Acad. Sci. USA,93: 9764–9769, 1996.

14. Brooks, P. C., Clark, R. A., and Cheresh, D. A. Requirement of vascular integrinavb3

for angiogenesis. Science (Washington DC),264: 569–571, 1994.15. Stromblad, S., Becker, J. C., Yebra, M., Brooks, P. C., and Cheresh, D. A. Suppres-

sion of p53 activity and p21WAF1/CIP1 expression by vascular cell integrinaVb3

during angiogenesis. J. Clin. Invest.,98: 426–433, 1996.16. Scatena, M., Almeida, M., Chaisson, M. L., Fausto, N., Nicosia, R. F., and Giachelli,

C. M. NFkB mediatesavb3 integrin-induced endothelial cell survival. J. Cell Biol.,141: 1083–1093, 1998.

17. Haimovitz-Friedman, A., Cordon-Cardo, C., Bayoumy, S., Garzotto, M.,McLoughlin, M., Gallily, R., Edwards, C. K., III, Schuchman, E. H., Fuks, Z., andKolesnick, R. Lipopolysaccharide induces disseminated endothelial apoptosisrequiring ceramide generation. J. Exp. Med.,186: 1831–1841, 1997.

18. Robaye, B., Mosselmans, R., Fiers, W., Dumont, J. E., and Galand, P. Tumor necrosisfactor induces apoptosis (programmed cell death) in normal endothelial cells in vitro.Am. J. Pathol.,138: 447–453, 1991.

19. Haimovitz-Friedman, A., Kan, C. C., Ehleiter, D., Persaud, R. S., McLoughlin, M.,Fuks, Z., and Kolesnick, R. N. Ionizing radiation acts on cellular membranes togenerate ceramide and initiate apoptosis. J. Exp. Med.,180: 525–535, 1994.

20. Araki, S., Shimada, Y., Kaji, K., and Hayashi, H. Apoptosis of vascular endothelialcells by fibroblast growth factor deprivation. Biochem. Biophys. Res. Commun.,168:1194–1200, 1990.

21. Meredith, J. E., Jr., Fazeli, B., and Schwartz, M. A. The extracellular matrix as a cellsurvival factor. Mol. Biol. Cell,4: 953–961, 1993.

22. Hannun, Y. A. Functions of ceramide in coordinating cellular responses to stress.Science (Washington DC),274: 1855–1859, 1996.

23. Verheij, M., Bose, R., Lin, X. H., Yao, B., Jarvis, W. D., Grant, S., Birrer, M. J.,Szabo, E., Zon, L. I., Kyriakis, J. M., Haimovitz-Friedman, A., Fuks, Z., andKolesnick, R. N. Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis. Nature (Lond.),380: 75–79, 1996.

24. Pena, L. A., Fuks, Z., and Kolesnick, R. Stress-induced apoptosis and the sphingo-myelin pathway. Biochem. Pharmacol.,53: 615–621, 1997.

25. Spiegel, S., Cuvillier, O., Edsall, L. C., Kohama, T., Menzeleev, R., Olah, Z., Olivera,A., Pirianov, G., Thomas, D. M., Tu, Z., Van Brocklyn, J. R., and Wang, F.Sphingosine-1-phosphate in cell growth and cell death. Ann. NY Acad. Sci.,845:11–18, 1998.

26. Reunanen, N., Westermarck, J., Hakkinen, L., Holmstrom, T. H., Elo, I., Eriksson,J. E., and Kahari, V. M. Enhancement of fibroblast collagenase (matrix metallopro-teinase-1) gene expression by ceramide is mediated by extracellular signal-regulatedand stress-activated protein kinase pathways. J. Biol. Chem.,273: 5137–5145, 1998.

27. Matthay, K. K., Harris, R., Reynolds, C. P., Shimada, H., Black, T., Stram, D. O., andSeeger, R. C. Improved event-free survival (EFS) for autologous bone marrowtransplantation (ABMT) vs. chemotherapy in neuroblastoma: a phase III randomizedChildren’s Cancer Group (CCG) study. Proc. Am. Soc. Clin. Oncol Annu. Meet.,17:525, 1998.

28. Reynolds, C. P., Villablanca, J. G., Stram, D. O., Harris, R., Seeger, R. C., andMatthay, K. K. 13-cis-retinoic acid given after intensive consolidation therapy forhigh risk neuroblastoma improves event-free survival (EFS): a randomized ChildrensCancer Group (CCG) study. Proc. Am. Soc. Clin. Oncol. Annu. Meet.,17: 2, 1998.

29. Stram, D. O., Matthay, K. K., O’Leary, M., Reynolds, C. P., Haase, G. M., Atkinson,J. B., Brodeur, G. M., and Seeger, R. C. Consolidation chemoradiotherapy andautologous bone marrow transplantation versus continued chemotherapy for meta-static neuroblastoma: a report of two concurrent Children’s Cancer Group studies.J. Clin. Oncol.,14: 2417–2426, 1996.

30. Meitar, D., Crawford, S. E., Rademaker, A. W., and Cohn, S. L. Tumor angiogenesiscorrelates with metastatic disease, N-mycamplification, and poor outcome in humanneuroblastoma. J. Clin. Oncol.,14: 405–414, 1996.

31. Sugiura, Y., Shimada, H., Seeger, R. C., Laug, W. E., and DeClerck, Y. A. Matrixmetalloproteinases-2 and -9 are expressed in human neuroblastoma: contribution ofstromal cells to their production and correlation with metastasis. Cancer Res.,58:2209–2216, 1998.

32. Ribatti, D., Alessandri, G., Vacca, A., Iurlaro, M., and Ponzoni, M. Human neuro-blastoma cells produce extracellular matrix-degrading enzymes, induce endothelialcell proliferation and are angiogenicin vivo. Int. J. Cancer,77: 449–454, 1998.

33. Wassberg, E., Pahlman, S., Westlin, J. E., and Christofferson, R. The angiogenesisinhibitor TNP-470 reduces the growth rate of human neuroblastoma in nude rats.Pediatr Res.,41: 327–333, 1997.

34. Nagabuchi, E., VanderKolk, W. E., Une, Y., and Ziegler, M. M. TNP-470 antian-giogenic therapy for advanced murine neuroblastoma. J. Pediatr. Surg.,32: 287–293,1997.

35. Lode, H. N., Moehler, T., Xiang, R., Jonczyk, A., Gillies, S. D., Cheresh, D. A., andReisfeld, R. A. Synergy between an antiangiogenic integrinav antagonist and anantibody-cytokine fusion protein eradicates spontaneous tumor metastases. Proc.Natl. Acad. Sci. USA,96: 1591–1596, 1999.

36. Stins, M. F., Prasadarao, N. V., Zhou, J., Arditi, M., and Kim, K. S. Bovine brainmicrovascular endothelial cells transfected with SV40-large T antigen: developmentof an immortalized cell line to study pathophysiology of CNS disease. In Vitro Cell.Dev. Biol., 33: 243–247, 1997.

37. Yatohgo, T., Izumi, M., Kashiwagi, H., and Hayashi, M. Novel purification ofvitronectin from human plasma by heparin affinity chromatography. Cell Struct.Funct.,13: 281–292, 1988.

38. Seeger, R. C., Atkinson, J., and Reynolds, C. P. Neuroblastoma.In: J. F. Holland, E.Frei III, R. C. Bast, Jr., D. W. Kufe, D. L. Morton, and R. R. Weichsenbaum (eds.),Cancer Medicine, pp. 2991–3020. Philadelphia: Lea and Febiger, 1996.

39. Shimada, H., Chatten, J., Newton, W. A., Jr., Sachs, N., Hamoudi, A. B., Chiba, T.,Marsden, H. B., and Misugi, K. Histopathologic prognostic factors in neuroblastictumors: definition of subtypes of ganglioneuroblastoma and an age-linked classifica-tion of neuroblastomas. J. Natl. Cancer Inst.,73: 405–416, 1984.

40. Gladson, C. L., and Cheresh, D. A. Glioblastoma expression of vitronectin and theavb3 integrin: adhesion mechanism for transformed glial cells. J. Clin. Invest.,88:1924–1932, 1991.

41. Lavie, Y., Cao, H., Bursten, S. L., Giuliano, A. E., and Cabot, M. C. Accumulationof glucosylceramides in multidrug-resistant cancer cells. J. Biol. Chem.,271:19530–19536, 1996.

42. Lavie, Y., Cao, H., Volner, A., Lucci, A., Han, T. Y., Geffen, V., Giuliano, A. E., andCabot, M. C. Agents that reverse multidrug resistance, tamoxifen, verapamil, andcyclosporin A, block glycosphingolipid metabolism by inhibiting ceramide glyco-sylation in human cancer cells. J. Biol. Chem.,272: 1682–1687, 1997.

43. Maurer, B. J., Metelitsa, L. S., Seeger, R. C., Cabot, M. C., and Reynolds, C. P.Increase of ceramide and induction of mixed apoptosis/necrosis byN-(4- hydroxy-phenyl)- retinamide in neuroblastoma cell lines. J. Natl. Cancer Inst.,91: 1138–1146,1999.

44. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: applicationto proliferation and cytotoxicity assays. J. Immunol. Methods,65: 55–63, 1983.

720

CERAMIDE IN INHIBITION OF ENDOTHELIAL INTEGRINS

on July 12, 2019. © 2000 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

45. Xu, J., Yeh, C. H., Chen, S., He, L., Sensi, S. L., Canzoniero, L. M. T., Choi, D. W.,and Hsu, C. Y. Involvement ofde novoceramide biosynthesis in tumor necrosisfactor-a/cycloheximide-induced cerebral endothelial cell death. J. Biol. Chem.,273:16521–16526, 1998.

46. Erdreich-Epstein, A., Liu, M., Liu, Y., and Durden, D. L. Protein tyrosine phospha-tase inhibitors in FcgRI-induced myeloid oxidant signaling. Exp. Cell Res.,237:288–295, 1997.

47. Durden, D. L., Kim, H. M., Calore, B., and Liu, Y. The FcgRI receptor signalsthrough the activation ofhckand MAP kinase. J. Immunol.,154: 4039–4047, 1995.

48. Re, F., Zanetti, A., Sironi, M., Polentarutti, N., Lanfrancone, L., Dejana, E., andColotta, F. Inhibition of anchorage-dependent cell spreading triggers apoptosis incultured human endothelial cells. J. Cell Biol.,127: 537–546, 1994.

49. Frisch, S. M., and Francis, H. Disruption of epithelial cell-matrix interactions inducesapoptosis. J. Cell Biol.,124: 619–626, 1994.

50. Gasparini, G., Brooks, P. C., Biganzoli, E., Vermeulen, P. B., Bonoldi, E., Dirix,L. Y., Ranieri, G., Miceli, R., and Cheresh, D. A. Vascular integrinavb3: a newprognostic indicator in breast cancer. Clin. Cancer Res.,4: 2625–2634, 1998.

51. Engels, K., Fox, S. B., and Harris, A. L. Angiogenesis as a biologic and prognosticindicator in human breast carcinoma. EXS,79: 113–156, 1997.

52. Okada, F., Rak, J. W., Croix, B. S., Lieubeau, B., Kaya, M., Roncari, L., Shirasawa,S., Sasazuki, T., and Kerbel, R. S. Impact of oncogenes in tumor angiogenesis: mutantK-ras up-regulation of vascular endothelial growth factor/vascular permeability factoris necessary, but not sufficient for tumorigenicity of human colorectal carcinomacells. Proc. Natl. Acad. Sci. USA,95: 3609–3614, 1998.

53. Montesano, R., Pepper, M. S., Mohle-Steinlein, U., Risau, W., Wagner, E. F., andOrci, L. Increased proteolytic activity is responsible for the aberrant morphogeneticbehavior of endothelial cells expressing the middle T oncogene. Cell,62: 435–445,1990.

54. Goodman, L. A., Liu, B. C., Thiele, C. J., Schmidt, M. L., Cohn, S. L., Yamashiro,J. M., Pai, D. S., Ikegaki, N., and Wada, R. K. Modulation of N-mycexpression altersthe invasiveness of neuroblastoma. Clin. Exp. Metastasis,15: 130–139, 1997.

55. Gladson, C. L., Hancock, S., Arnold, M. M., Faye Petersen, O. M., Castleberry, R. P.,and Kelly, D. R. Stage-specific expression of integrinavb3 in neuroblastic tumors.Am. J. Pathol.,148: 1423–1434, 1996.

56. Kemperman, H., Wijnands, Y. M., and Roos, E.aV Integrins on HT-29 coloncarcinoma cells: adhesion to fibronectin is mediated solely by small amounts ofaVb6,andaVb5 is codistributed with actin fibers. Exp. Cell Res.,234: 156–164, 1997.

57. Lehmann, M., Rabenandrasana, C., Tamura, R., Lissitzky, J. C., Quaranta, V., Pichon,J., and Marvaldi, J. A monoclonal antibody inhibits adhesion to fibronectin andvitronectin of a colon carcinoma cell line and recognizes the integrinsavb3, avb5, andavb6. Cancer Res.,54: 2102–2107, 1994.

58. Nishimura, S. L., Boylen, K. P., Einheber, S., Milner, T. A., Ramos, D. M., andPytela, R. Synaptic and glial localization of the integrinavb8 in mouse and rat brain.Brain Res.,791: 271–282, 1998.

59. Cheresh, D. A., and Spiro, R. C. Biosynthetic and functional properties of anArg-Gly-Asp-directed receptor involved in human melanoma cell attachment tovitronectin, fibrinogen, and von Willebrand factor. J. Biol. Chem.,262: 17703–17711, 1987.

60. Zundel, W., and Giaccia, A. Inhibition of the anti-apoptotic PI(3)K/Akt/Bad pathwayby stress. Genes Dev.,12: 1941–1946, 1998.

61. Chen, C. S., Mrksich, M., Huang, S., Whitesides, G. M., and Ingber, D. E. Geometriccontrol of cell life and death. Science (Washington DC),276: 1425–1428, 1997.

62. Buckley, C. D., Pilling, D., Henriquez, N. V., Parsonage, G., Threlfall, K., Scheel-Toellner, D., Simmons, D. L., Akbar, A. N., Lord, J. M., and Salmon, M. RGDpeptides induce apoptosis by direct caspase-3 activation. Nature (Lond.),397: 534–539, 1999.

63. Soldi, R., Mitola, S., Strasly, M., Defilippi, P., Tarone, G., and Bussolino, F. Role ofavb3 integrin in the activation of vascular endothelial growth factor receptor-2.EMBO J.,18: 882–892, 1999.

64. Brenner, B., Grassme, H. U., Muller, C., Lang, F., Speer, C. P., and Gulbins, E.L-selectin stimulates the neutral sphingomyelinase and induces release of ceramide.Exp. Cell Res.,243: 123–128, 1998.

65. Brenner, B., Koppenhoefer, U., Weinstock, C., Linderkamp, O., Lang, F., andGulbins, E. Fas- or ceramide-induced apoptosis is mediated by a Rac1-regulatedactivation of Jun N-terminal kinase/p38 kinases and GADD153. J. Biol. Chem.,272:22173–22181, 1997.

66. Frisch, S. M., Vuori, K., Kelaita, D., and Sicks, S. A role for Jun-N-terminal kinasein anoikis: suppression by bcl-2 and crmA. J. Cell Biol.,135: 1377–1382, 1996.

67. Ameyar, M., Atfi, A., Cai, Z., Stancou, R., Shatrov, V., Bettaieb, A., and Chouaib, S.Analysis of human breast adenocarcinoma MCF7 resistance to tumor necrosis factor-induced cell death: lack of correlation between JNK activation and ceramide pathway.J. Biol. Chem.,273: 29002–29008, 1998.

68. Khwaja, A., and Downward, J. Lack of correlation between activation of Jun-NH2-terminal kinase and induction of apoptosis after detachment of epithelial cells. J. CellBiol., 139: 1017–1023, 1997.

69. Modur, V., Zimmerman, G. A., Prescott, S. M., and McIntyre, T. M. Endothelial cellinflammatory responses to tumor necrosis factor-a: ceramide-dependent and -inde-pendent mitogen-activated protein kinase cascades. J. Biol. Chem.,271: 13094–13102, 1996.

70. Adam, D., Ruff, A., Strelow, A., Wiegmann, K., and Kronke, M. Induction ofstress-activated protein kinases/c-Jun N-terminal kinases by the p55 tumour necrosisfactor receptor does not require sphingomyelinases. Biochem. J.,333:343–350, 1998.

71. Zhu, P., Xiong, W., Rodgers, G., and Qwarnstrom, E. E. Regulation of interleukin 1signalling through integrin binding and actin reorganization: disparate effects onNFkB and stress kinase pathways. Biochem. J.,330: 975–981, 1998.

72. Westwick, J. K., Bielawska, A. E., Dbaibo, G., Hannun, Y. A., and Brenner, D. A.Ceramide activates the stress-activated protein kinases. J. Biol. Chem.,270: 22689–22692, 1995.

73. Dbaibo, G. S., Obeid, L. M., and Hannun, Y. A. Tumor necrosis factor-a (TNF-a)signal transduction through ceramide: dissociation of growth inhibitory effects ofTNF-a from activation of nuclear factor-kB. J. Biol. Chem.,268: 17762–17766,1993.

74. Boland, M. P., and O’Neill, L. A. Ceramide activates NFkB by inducing theprocessing of p105. J. Biol. Chem.,273: 15494–15500, 1998.

75. Zhou, H., Summers, S. A., Birnbaum, M. J., and Pittman, R. N. Inhibition of Aktkinase by cell-permeable ceramide and its implications for ceramide-induced apop-tosis. J. Biol. Chem.,273: 16568–16575, 1998.

76. Spinedi, A., Amendola, A., Di Bartolomeo, S., and Piacentini, M. Ceramide-inducedapoptosis is mediated by caspase activation independently from retinoblastoma pro-tein post-translational modification. Biochem. Biophys. Res. Commun.,243: 852–857, 1998.

77. Spinedi, A., Di Bartolomeo, S., Di Sano, F., Rodolfo, C., Ambrosino, A., andPiacentini, M. Ceramide accumulation precedes caspase-dependent apoptosis in CHP-100 neuroepithelioma cells exposed to the protein phosphatase inhibitor okadaic acid.Cell Death Differ.,6: 618–623, 1999.

78. Kolesnick, R. N., and Kronke, M. Regulation of ceramide production and apoptosis.Annu. Rev. Physiol.,60: 643–665, 1998.

79. Mathias, S., Pena, L. A., and Kolesnick, R. N. Signal transduction of stress viaceramide. Biochem. J.,335: 465–480, 1998.

80. Dbaibo, G. S., Perry, D. K., Gamard, C. J., Platt, R., Poirier, G. G., Obeid, L. M., andHannun, Y. A. Cytokine response modifier A (CrmA) inhibits ceramide formation inresponse to tumor necrosis factor (TNF)-a: CrmA and Bcl-2 target distinct compo-nents in the apoptotic pathway. J. Exp. Med.,185: 481–490, 1997.

81. Tepper, A. D., de Vries, E., van Blitterswijk, W. J., and Borst, J. Ordering of ceramideformation, caspase activation, and mitochondrial changes during CD95- and DNAdamage-induced apoptosis. J. Clin. Invest.,103: 971–978, 1999.

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2000;60:712-721. Cancer Res   Anat Erdreich-Epstein, Hiroyuki Shimada, Susan Groshen, et al.   with Increased Endogenous Ceramide High-Risk Neuroblastoma and Their Inhibition Is Associated

Are Expressed by Endothelium of5βvα and 3βvαIntegrins

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